G proteins and their receptors play a key controlling and decisive role in regulating cellular physiology [1]. Some of the regulatory signaling pathways mediated by receptors and G proteins are implicated in the onset and progression of serious and fatal human diseases. G proteins comprise an α subunit and a βγ subunit complex. G proteins are signal transducers—that is they mediate the conversion of an extracellular signal into an intracellular physiological response. On sensing a hormone, neurotransmitter, a natural or chemically synthesized agonist, an excited receptor activates a G protein resulting in the dissociation of the α subunit and βγ complex which subsequently regulate the function of effectors inside the cell. (See also BIOCHEMISTRY, Third Edition, Lubert Stryer, W.H. Freeman and Company, N.Y., in particular Chapter 38 thereof, including page 979).
In live mammalian systems such as human, rat and mice, G protein signaling pathways are extraordinarily complex compared to G protein signaling pathways in single cell organisms such as yeast (Saccharomyces cerevisiae) and soil amoeba (Dictyostelium discoideum). Yeast and soil amoeba cells contain a few G protein coupled receptor types and G protein types while in contrast mammalian cells contain hundreds of G protein coupled receptor types and a large variety of G protein subunit types [2].
Many of the molecular mechanisms underlying G protein signaling pathways have so far been elucidated in in vitro systems using purified proteins and broken cells. However, G protein signaling operations occur in intact living cells subject to constraints of dynamic equilibrium, which are disrupted when cells are broken.
Additionally, as mentioned before, mammalian cells contain large families of G protein subunits, receptors and effector molecules leading to the generation of vast networks of membrane transduction signaling pathways which are functional only when the cell is intact and living [3]. Unfortunately, relatively little information is at present available about the behavior of these signaling pathways in an intact living mammalian cell because methods have not been available for their observation.
Several mechanisms at the basis of G protein signaling have been identified so far. Results have shown that receptor stimulated dissociation of the G protein subunits leads to the activation of effectors downstream and thus signaling pathways. Both activated subunits, the GTP bound α subunit and the βγ complex, act on effector molecules [4]. Subsequent formation of the G protein heterotrimer as a result of receptor inactivation, switches off effector signaling activity of the G protein subunits. In order to elucidate more information, soil amoeba (D. discoideum) G protein subunits have been labeled with fluorescent proteins and expressed in soil amoeba (D. discoideum) cells providing the capability of detecting a FRET signal emanating from a heterotrimer and detecting the loss of FRET signal upon activation and subsequent dissociation of the heterotrimer [5].
G protein coupled receptors form the single largest target for commercially available pharmaceutical drugs today. It is estimated that fifty percent of recently launched drugs were targeted at these receptors with annual worldwide sales exceeding about $30 billion in year 2001. Among the one hundred highest selling drugs, about 25% were directed at G protein coupled receptors [6].
However, today's available commercial drugs are targeted at a relatively small proportion of known G protein coupled receptors.
While the three dimensional structure of the G protein coupled receptor and newer methods of rational drug design increase the range and depth of candidate molecules are available, there is at present an undesired serious limitation in methods available to screen drug candidates non-invasively using mammalian G protein coupled receptors and G proteins.
There is also a lack of information about the temporal changes and spatial localization of the effects of candidate therapeutic molecules in an intact living cell.